How does SnapGene predict fidelity in Golden Gate Cloning simulation?
SnapGene 6.1 and later provides a dedicated Golden Gate Assembly tool for automated design of PCR primers with appropriately positioned Type IIS restriction sites (when required), and for simulation of Golden Gate Assembly. See the lessons in the Golden Gate Assembly chapter of this user guide to learn how to use the Golden Gate tool.
The SnapGene Golden Gate tool can be used to simulate circular assembly of up to 100 fragments.
SnapGene uses empirical data from Potapov etal. (2018) to choose compatible combinations of unique nucleotide overhangs for Golden Gate Assembly†.
SnapGene uses this data to calculate predicted ligation fidelity for individual overhangs (an estimate of the potential for mis-ligation to other overhangs present in the reaction).
SnapGene also predicts overall "Assembly fidelity". Assembly fidelity is calculated by multiplying the predicted ligation fidelity of all unique overhangs used for assembly.
† Overhang length will depend on the Type IIS restriction enzyme used for the Golden Gate reaction. SnapGene can estimate fidelity for assemblies that use 3 or 4 nucleotide overhangs. SnapGene cannot estimate fidelity for Type IIS enzymes that create 1, 2 or 5+ nucleotide overhangs. For 3- or 4-base overhangs, SnapGene uses the Potapov etal. (2018) dataset for T4 DNA ligase, 18 hours at 25°C.
When Golden Gate assembly is ready to proceed, predicted assembly fidelity is reported in the lower right panel of the Golden Gate Assembly Window.
In the above example, using the given set of overhangs (TATG, AGAT, TGGT), Golden Gate Assembly is predicted to yield 99.9% of correctly-ligated products.
To view predicted fidelity for individual overhangs (in the context of all other overhangs), once Golden Gate assembly is ready to proceed, click Adjust Overhangs... and use the "Overhang" dropdown to view the predicted ligation fidelity for each alternative overhang that could be used for assembly.